Quant monochromatic microplate spectrophotometer

Crystal violet staining was performed to assess cell viability. Cells were incubated in 48-well plates at 2.0 × 10⁴ cells per well and cell viability was measured at the indicated time points. For crystal violet staining, the culture medium was removed; the cells were washed with PBS and fixed with 4% formaldehyde in PBS at room temperature for 10 min, washed with PBS and stained with 0.1% crystal violet for 30 min at room temperature. The cells were then washed with water, after which the water was removed and the cells were dried out. Crystal violet dye was dissolved in 200 μl of 10% acid acetic and transferred to 96-well plate. Optical densities were measured at a wavelength of 590 nm using a Bio-tek µQuant Monochromatic Microplate Spectrophotometer. Experiments were performed as independent biological triplicates in technical duplicates and data are presented as mean ± SD.

Aliquots (10 µL) of the TCA-precipitated protein sample taken after adding TCA in the second round were used to measure the total protein contents. Three technical replicates of each biological replicate were used to determine the total amount of protein per biological replicate. The amount was quantified using the BioRad RC/DC kit (Bio-Rad, Sundbyberg, Sweden) according to the manufacturer’s instructions. Spectrophotometric measurements were made using a µQuant monochromatic microplate spectrophotometer (BioTek, Winooski, VT, USA).